2.1. The Human Immunodeficiency Virus
The primary cause of acquired immunodeficiency syndrome (AIDS) has been shown to be HIV (Barre-Sinoussi et al., 1983, Science 220:868-870; Gallo et al., 1984, Science 224:500-503). HIV causes immunodeficiency in an individual by infecting important cell types of the immune system, which results in their depletion. This, in turn, leads to opportunistic infections, neurological dysfunctions, neoplastic growth, and death.
HIV is a member of the lentivirus family of retroviruses (Teich et al., 1984, RNA TUMOR VIRUSES, Weiss et al., eds., CSH-Press, pp. 949-956). Retroviruses are small enveloped viruses that contain a diploid, single-stranded RNA genome, and replicate via a DNA intermediate produced by a virally-encoded reverse transcriptase, an RNA-dependent DNA polymerase (Varmus, 1988, Science 240:1427-1439). There are at least two distinct subtypes of HIV: HIV-1 (Barre-Sinoussi et al., 1983, Science 220:868-870; Gallo et al., 1984, Science 224:500-503) and HIV-2 (Clavel et al., 1986, Science 233:343-346; Guyader et al., 1987, Nature 326:662-669). Genetic heterogeneity exists within each of these HIV subtypes.
CD4.sup.+ T cells are the major targets of HIV infection because the CD4 cell surface protein acts as a cellular receptor for HIV attachment (Dalgleish et al., 1984, Nature 312:763-767; Klatzmann et al., 1984, Nature 312:767-768; Maddon et al., 1986, Cell 47:333-348). Viral entry into cells is dependent upon viral protein gp 120 binding to the cellular CD4 receptor molecule (McDougal et al., 1986, Science 231:382-385; Maddon et al., 1986, Cell 47:333-348), and a chemokine co-receptor such as CXCR4 or CCR5 (Moore, 1997, Science 276:51-52).
2.2. HIV Treatment
HIV infection is pandemic and HIV-associated diseases have become a world-wide health problem. Despite considerable efforts in the design of anti-HIV modalities, there is, thus far, no successful prophylactic or therapeutic regimen against AIDS. However, several stages of the HIV life cycle have been considered as potential targets for therapeutic intervention (Mitsuya et al., 1991, FASEB J. 5:2369-2381). For example, virally-encoded reverse transcriptase has been a major focus of drug development. A number of reverse-transcriptase-targeted drugs, including 2', 3'-dideoxynucleotide analogs such as AZT, ddI, ddC, and ddT have been shown to be active against HIV (Mitsuya et al., 1990, Science 249:1533-1544). While beneficial, these nucleotide analogs are not curative, probably due to the rapid appearance of drug resistant HIV mutants (Lander et al., 1989, Science 243:1731-1734). In addition, the drugs often exhibit toxic side effects, such as bone marrow suppression, vomiting, and liver abnormalities.
Another stage of the HIV life cycle that has been targeted is viral entry into the cells, the earliest stage of HIV infection. This approach has primarily utilized recombinant soluble CD4 protein to inhibit infection of CD4.sup.30 T cells by some HIV-1 strains (Smith et al., 1987, Science 238:1704-1707). Certain primary HIV-1 isolates, however, are relatively less sensitive to inhibition by recombinant CD4 (Daar et al., 1990, Proc. Natl. Acad. Sci. USA 87:6574-6579). To date, recombinant soluble CD4 clinical trials have produced inconclusive results (Schooley et al., 1990, Ann. Int. Med. 112:247-253; Kahn et al., 1990, Ann. Int. Med. 112:254-261; Yarchoan et al., 1989, Proc. Vth Int. Conf. on AIDS, p. 564, MCP 137).
Additionally, the later stages of HIV replication which involve crucial virus-specific secondary processing of certain viral proteins and enzymes have also been targeted for anti-HIV drug development. Late stage processing is dependent on the activity of a virally-encoded protease, and drugs including nelfinavir, saquinavir, ritonavir, and indinavir have been developed to inhibit this protease (Pettit et al., 1993, Persp. Drug. Discov. Design 1:69-83). With this class of drugs, the emergence of drug resistant HIV mutants is also a problem; resistance to one inhibitor often confers cross resistance to other protease inhibitors (Condra et al., 1995, Nature 374:569-571). These drugs often exhibit toxic side effects such as nausea, altered taste, circumoral parethesias, lipodystrophy and nephrolithiasis. Furthermore, these drugs also interact with many drugs commonly used to treat HIV infection (Flexner, 1998, N. Engl. J. Med. 338:1281-1292).
Antiviral therapy of HIV using different combinations of nucleoside analogs and protease inhibitors have recently been shown to be more effective than the use of a single drug alone (Torres et al., 1997, Infec. Med. 14:142-160). However, despite the ability to achieve significant decreases in viral burden, there is no evidence to date that combinations of available drugs will afford a curative treatment for AIDS.
Other approaches for developing treatment for AIDS include the delivery of exogenous genes into infected cells. One such gene therapy approach involves the use of genetically-engineered viral vectors to introduce toxic gene products to kill HIV-infected cells. For instance, replication defective vectors have been designed to introduce cell growth inhibitory genes into host cells (WO 90/12087, Oct. 18, 1980). One strategy attempted by several groups involves the delivery of the herpes simplex virus type 1 thymidine kinase (tk) toxin gene. The tk gene product is toxic to mammalian cells only in the presence of nucleoside analogs, such as ganciclovir (Ventakash et al., 1990, Proc. Natl. Acad. Sci. USA 87: 8746-8750; Brady et al., 1994, Proc. Natl. Acad. Sci. USA 91: 365-369; WO 90/07936, Jul. 26, 1990). Diphtheria toxin gene has also been used, and the gene was placed under the control of cis-acting HIV regulatory sequences (U.S. Pat. No. 5,306,631, issued Apr. 26, 1994). Others have utilized replication incompetent mutants of HIV which have the potential to express an inhibitory gene product in the presence of HIV tat (WO 94/16060, Jul. 21, 1994).
Another form of gene therapy is designed to protect virally-infected cells from cytolysis by specifically disrupting viral replication. Efforts to identify appropriate protective genes have, in large part, been based on an understanding of the molecular biology of HIV replication. A few examples of this approach are as follows.
The HIV-1 Rev gene encodes a protein that is necessary for the expression of full length HIV-1 transcripts in infected cells and the production of HIV-1 virions. Transfection with one Rev mutant known as RevM10 has been shown to protect cells against HIV infection (Malim et al., 1992, J. Exp. Med. 176:1197; Bevec et al., 1992, Proc. Natl. Acad. Sci. USA 89:9870-74). Typically, the transfectants are resistant to HIV-1 infection for about 2 weeks from the time of inoculation before resistant variants appear (Woffendin et al., 1994, Proc. Natl. Acad. Sci. USA 91: 11581-85).
In addition, Rev function can be interfered with by producing an excess of the binding site of the Rev protein, termed Rev Response Element (RRE), which prevents the binding of Rev to RRE of viral transcripts. A "decoy" which consisted of achimeric RNA composed of an RRE and a tRNA prevented infection of cultured cells for a period of greater than about 40 days (Lee et al., 1994, J. Virol. 68:8254-64).
Alternatively, fusion proteins capable of binding to viral env proteins have been made to prevent the production of HIV-1 virions. Examples include a fusion protein composed of CD4 and a lysosomal targeting protein procathepsin D, and an anti-env Fv which is secreted into the endoplasmic reticulum (Lin et al., WO 93/06216; Marasco et al., 1993, Proc. Natl. Acad. Sci. USA 90:7889-93).
Antisense polynucleotides have also been designed to complex with and sequester the HIV-1 transcripts (Holmes et al., WO 93/11230; Lipps et al., WO 94/10302; Kretschmer et al., EP594,881; and Chatterjee et al., 1992, Science 258:1485). Furthermore, an enzymatically active RNA, termed ribozyme, has been used to cleave viral transcripts. The ribozyme approach to forming an HIV-1 resistant hematopoietic cell line has been reported (Ojwang et al., 1992, Proc. Natl. Acad. Sci. USA 89:10802-06; Yamada et al., 1994, Gene Therapy 1:38-45; Ho et al., WO94/26877; and Cech and Sullenger, WO 95/13379).
Roninson et al. described a method for isolating genetic fragments from the HIV-1 genome capable of protecting a cell from HIV-1 infection (U.S. Pat. No. 5,217,889 and WO 92/07071). The method involves the preparation of an expression library known as a Random Fragment Expression (RFE) library that contains random sequence fragments of the HIV-1 genome. Gene fragments referred to as HIV-1 Genetic Suppressor Element (HIV-1 GSE) are then selected from the RFE library following an extensive selection procedure. The selection step involves transfection of the RFE library into a cell line to which HIV-1 infection is normally cytotoxic. However, the low sensitivity of this selection step greatly limits the practical use of the procedure. Moreover, no specific GSE sequences were reported using this method that were capable of suppressing HIV-1 infection.
2.3. Tumorigenesis
It has been reported that overexpression of the hdm2 protein drives oncogenesis through antagonism of the p53 tumor suppressor protein. Binding of hdm2 to p53 promotes the degradation of p53. Several recent reports focus on the requirement for the hdm2 protein to actively shuttle between nucleus and cytoplasm in order to exert inhibition of p53 (Lain et al., 1999, Exp. Cell Res. 248(2):457-472). An inhibitor of nuclear expert activates the p53 response and induces the localization of HDM2 and p53 to U1A-positive nuclear bodies associated with the PODs (Roth et al., 1998, EMBO J. 15:554-564). Nucleo-cytoplasmic shuttling of the hdm2 oncoprotein regulates the levels of the p53 protein via a pathway used by the human immunodeficiency virus rev protein (Tao et al., 1999, Proc. Natl. Acad. Sci. USA 96:3077-3080). P19(ARF) stabilizes p53 by blocking nucleo-cytoplasmic shuttling of Mdm2 (Tao et al., 1999, Proc. Natl. Acad. Sci. USA 96:6937-6941). Thus, shuttling of the hdm2 protein apparently depends on a nuclear export pathway that overlaps, or is identical to, that utilized by the HIV rev protein. Furthermore, the p19(ARF) tumor suppressor stabilizes p53, thereby inhibiting tumorigenesis, by interfering with this nucleo-cytoplasmic shuttling.
Thus, there remains a need to isolate and identify genetic suppressor elements that are involved in the inhibition of HIV infection or tumorigenesis.